FlV causes an AIDS-like syndrome in the domestic cat and represents the smallest natural animal system for study of lentivirus infections. The protease (PR) shares substantial structural homology with PR of HIV-1 but has unique substrate and inhibitor properties, some of which are shared with PRs of drug-resistant HIVs. Studies to date have identified critical amino acid residues of FlV and HIV-1 PRs that are responsible for the observed substrate/inhibitor specificities and mutant FJVs have been prepared that show HIV-like substrate/inhibitor specificities. This information has been used to develop a broad-based inhibitor (termed TL-3) that is efficacious against both FlV and HIV-l PRs in vitro and virus infection in tissue culture. We propose to further define substrate/inhibitor interactions by identifying structural changes responsible for escape by drug-resistant HIV- I and FIV mutants and establishing the limits of protease mutability. Initial experiments have identified a mutant HIV with a 19-fold increase in resistance to TL-3. The PR of this isolate as well as wild-type and other drug resistant PRs have been sub-cloned, over-expressed in E. coli, and purified for enzymatic analyses. These PRs will be employed in conjunction with phage display libraries to select for substrates preferred by drug resistant variant PRs and optimal cleavage sequences will be identified. The information gained can be used to prepare inhibitors with greater selectivity for drug-resistant PRs. Consensus sequences will be identified that represent optimal cleavage targets for a broad spectrum of F1V and HIV-l PRs, which can be used as frameworks to prepare broad-based inhibitors efficacious against a wide range of FIV and HIV variants. Tests will be performed in tissue culture and in vivo against both FIV (in cats) and HIV-1 (in NOD/SCID mice) to monitor inhibitor efficacy and to test for relative viral fitness and development of drug resistance. These studies will advance our understanding of the structural basis for drug resistance and lead to development of effective inhibitors less susceptible to escape via mutations within PR.